Intestinal nontuberculous mycobacteria and environmental enteropathy in Zambia: hospital-based observations.

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Chongwe, Gershom
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The University of Zambia
Background Environmental enteropathy is prevalent in many low and middle-income countries. Although its aetiology is unknown, we hypothesised that nontuberculous mycobacteria (NTMs), which are ubiquitous in the environment and are known to cause disease in the lungs, the gut, skin, bloodstream and joints, could play a role in pathogenesis. We estimated the prevalence of intestinal carriage of NTMs and investigated whether Mycobacterium avium antigens could contribute to environmental enteropathy through matrix metalloproteinase (MMP)-mediated intestinal damage. Methods This was a sequential multimethod design comprising a hospital-based cross sectional study as phase one followed by a quasi-experimental post-test study as phase two. In the first phase, 97 patients scheduled for routine endoscopy were surveyed to determine the prevalence of NTM in the gut. Stool, intestinal lavage samples, and biopsy samples from the descending colon and caecum were collected. The samples were analysed using the Mycobacteria Growth Indicator Tube (MGIT) liquid culture method. In the second phase of the study, 48 participants were recruited for an in-vitro study to determine gut immune responses to Mycobacterium avium. Small intestinal biopsies and whole blood samples were stimulated with M. avium lysate over 24 hours. Unstimulated biopsy or blood samples served as negative controls, while stimulation with Staphylococcal enterotoxin B served as a positive control. Supernatants were used to quantify MMP-1, -2, -8 and -9 expression using ELISA, and interleukin 17A (IL-17A), IL-10, IL-6, IL-4, IL-2, IL-1β, interferon (IFN) gamma and tumour necrosis factor (TNF) alpha by flow cytometric assay. Questionnaires were used to collect demographic and clinical information in both phases. Results From the survey, out of the 97 patients, 52 (53.6%) were males. The mean age was 46.6 (±15.9), range (18–80) years. The prevalence of NTM was 7.2% (95% CI 1.9–12.4), while that of Mycobacterium tuberculosis (MTB) was 6.2% (95% CI 2.3–13.0). Carriage of NTM was not significantly associated with age, sex or presenting symptoms such as diarrhoea, abdominal pain, weight loss as well as HIV status. Descending colon samples were the most likely to be positive (9.8%, 95% CI 3.7, 15.8) followed by stool samples (6.8%, 95% CI 1.0–12.6), caecal biopsy (6.1%, 95% CI 0.3–11.8) and intestinal lavage samples (5.9%, 95% CI 0.3– vii 11.5). In vitro experiments using duodenal biopsies from 48 patients (21 men, 27 women, median age 35 years) demonstrated that M. avium lysate induced the expression of many Th1, Th2 and Th17 cytokines in peripheral blood but only IL-1β and IL-6 in duodenal tissue. M. avium lysate induced the expression of MMP-1 in duodenal tissue (p=0.004) compared to negative controls, but expression of MMPs 2, 8, or 9 were not significantly increased. Discussion and Conclusion These results have revealed a prevalence of NTMs of seven percent in this population, suggesting an environmental contamination of the gut by potentially pathogenic NTMs that was not associated with any symptoms or demographic status. We have also shown that M. avium induced the expression of MMP-1 in duodenal tissue and in peripheral blood. M. avium also induced the expression of a restricted set of cytokines in duodenal tissue, namely IL-1β and IL-6 as well as eliciting a Th1 and Th2 response in the blood. These findings suggest that NTM can no longer be dismissed as mere contaminants during investigations for other diseases. We speculate that the induction of these molecules by M. avium suggests a possible pathway through which NTMs, and M. avium in particular, could remodel the intestinal mucosa and lead to environmental enteropathy. As techniques for isolation of these organisms improve, coupled with a deeper understanding of the scope of disease caused by the organisms, the case can be made for mechanisms to improve both surveillance and diagnostic capacity in resource-poor settings.
Thesis of Doctor of Philosophy in Epidemiology